Shiga toxins (Stxs, verotoxins) are proteinous exotoxins produced by enterohemorrhagic Escherichia coli of pathogenic Escherichia coli species. The Shiga toxins cause hemorrhagic enteritis, hemolytic uremic syndrome, encephalopathy, and the like.
The Shiga toxins are broadly classified into Stx1 and Stx2, each of which is further classified into subclasses. An example of the Stx2 includes Stx2e which causes swine edema disease. The swine edema disease is known to frequently occur in baby pigs one to two weeks after weaning. A fatality due to infection with edema disease bacteria is 50 to 90%, which is extremely high.
Further, cholera toxins (CTs) are proteinous exotoxins produced by Vibrio cholerae. The CTs are known to cause severe diarrhea and emesis.
Still further, Escherichia coli heat-labile toxins (LTs) are proteinous endotoxins produced by enterotoxigenic Escherichia coli. The LTs are known to cause diarrhea and emesis.
The bacterial toxins, Stxs, LTs, and CTs are all known to include a B-subunit pentamer involved in adhesion to cells and an A-subunit monomer having a toxicity. The LTs and the CTs are also known to be similar structurally and functionally.
As a method of preventing the diseases caused by those bacterial toxins, methods of administering a vaccine by an injection or a nasal spray and administering the vaccine orally are known.
For example, a technology where an attenuated Stx2e protein is produced using recombinant Escherichia coli and administered to pigs by an injection is known (Non-patent Document 1). However, for example, an amount of the attenuated Stx2e protein produced by the recombinant Escherichia coli is not sufficient and the administration of the vaccine by an injection requires human labor. This has been a problem.
Further, the method of administering the vaccine orally draws increasing attention in terms of reducing the labor in a stockbreeding field. In such a context, a technology where the bacterial toxin protein is produced by plants using a transgenic technology has been being developed. For example, a transgenic plant containing DNA encoding an LT protein B subunit (LTB) and expressing the DNA has been described (Patent Documents 1 and 2). A transgenic plant expressing DNA encoding the LT protein or the CT protein has been also described (Patent Document 3). However, there has been a problem that the amount of the produced protein is not sufficient in those technologies. An example of producing the LTB in Lactuca sativa has been reported (Non-patent Document 2). In this study, a gene of the LT protein B subunit including modified codons is expressed in Lactuca sativa using both a cauliflower mosaic virus 35S RNA promoter (CaMV35S) which is a promoter expressed highly in a plant and Kozak sequence which is an enhancer. As a result, it has been reported that the LT protein B subunit is accumulated in an amount of about 2.0% by mass of a total soluble protein of Lactuca sativa. However, this extent of the accumulated protein is thought to be insufficient to efficiently prevent a bacterial disease by utilizing the transgenic plant. That is, it is necessary to efficiently produce and accumulate the target bacterial toxin protein in plant cells.
The inventors of the present invention found that the Stx2e protein could be produced efficiently in a plant such as Lactuca sativa and accumulated at a high concentration in a plant body by expressing the Stx2e protein where a secretory signal peptide derived from a plant had been added to its amino terminus, using a 5′-untranslated region (ADH 5′-UTR) of an alcohol dehydrogenase gene derived from a plant, and filed the patent (Patent Document 4).
[Patent Document 1] JP 10-507916 A    [Patent Document 2] JP 2000-166411 A    [Patent Document 3] JP 2002-533068 A    [Patent Document 4] WO 2009/004842 A1    [Non-patent Document 1] Makino et al., Microbial Pathogenesis, Volume 31, Number 1, Jul. 2001, pp. 1-8(08)    [Non-patent Document 2] Kim et al., Protein Expression and Purification, Volume 51, Number 1, Jan. 2006, pp. 22-27(06)